scholarly journals Individual Somatic H1 Subtypes Are Dispensable for Mouse Development Even in Mice Lacking the H10Replacement Subtype

2001 ◽  
Vol 21 (23) ◽  
pp. 7933-7943 ◽  
Author(s):  
Yuhong Fan ◽  
Allen Sirotkin ◽  
Robert G. Russell ◽  
Julianna Ayala ◽  
Arthur I. Skoultchi
Keyword(s):  
Knockout Mice ◽  
Knockout Mouse ◽  
Embryonic Stem ◽  
Mouse Development ◽  
Double Knockout ◽  
Linker Histones ◽  
Deficient Mice ◽  
Mouse Lines

ABSTRACT H1 linker histones are involved in facilitating the folding of chromatin into a 30-nm fiber. Mice contain eight H1 subtypes that differ in amino acid sequence and expression during development. Previous work showed that mice lacking H10, the most divergent subtype, develop normally. Examination of chromatin in H10−/− mice showed that other H1s, especially H1c, H1d, and H1e, compensate for the loss of H10 to maintain a normal H1-to-nucleosome stoichiometry, even in tissues that normally contain abundant amounts of H10 (A. M. Sirotkin et al., Proc. Natl. Acad. Sci. USA 92:6434–6438, 1995). To further investigate the in vivo role of individual mammalian H1s in development, we generated mice lacking H1c, H1d, or H1e by homologous recombination in mouse embryonic stem cells. Mice lacking any one of these H1 subtypes grew and reproduced normally and did not exhibit any obvious phenotype. To determine whether one of these H1s, in particular, was responsible for the compensation present in H10−/− mice, each of the three H1 knockout mouse lines was bred with H10 knockout mice to generate H1c/H10, H1d/H10, or H1e/H10double-knockout mice. Each of these doubly H1-deficient mice also was fertile and exhibited no anatomic or histological abnormalities. Chromatin from the three double-knockout strains showed no significant change in the ratio of total H1 to nucleosomes. These results suggest that any individual H1 subtype is dispensable for mouse development and that loss of even two subtypes is tolerated if a normal H1-to-nucleosome stoichiometry is maintained. Multiple compound H1 knockouts will probably be needed to disrupt the compensation within this multigene family.

A Novel Role of Coagulation Proteases on Viral-Based Gene Transfer Efficacy.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 691-691
Author(s):  
Joerg Schuettrumpf ◽  
Jianxiang Zou ◽  
Shin Jen Tai ◽  
Alexander Schlachterman ◽  
Kian Tian ◽  
...  
Keyword(s):  
Gene Expression ◽  
Gene Transfer ◽  
Knockout Mice ◽  
Viral Vectors ◽  
Hemophilia B ◽  
Gene Copy ◽  
Dependent Manner ◽  
Deficient Mice

Abstract Coagulation proteases are crucial for hemostasis and have also been implicated in inflammatory responses, blood vessel formation, and tumor cell metastasis. Cellular responses triggered by proteases are mediated by protease-activated receptors (PAR). Adeno-associated virus (AAV)-2 vectors hold promise for the treatment of several diseases and were already tested in Phase I studies for hemophilia B following intramuscular or hepatic artery deliveries. Previously, we determined an unexpected inhibitory effect (60–70% downregulation) on AAV-2 and adenovirus mediated gene transfer by thrombin- or FXa inhibitors. These results were independent of mouse strain, transgene product, or vector promoter, and gene expression by vectors of alternate serotypes AAV-5 or -8, which do not share cellular receptors with AAV-2, were not affected by any drug. Here we present in vivo evidence of a novel role of coagulation proteases and PARs in modulating gene transfer by viral vectors. We tested AAV-2 gene transfer efficacy in (a) animal models for proteases deficiency [FX and FIX deficient animals], (b) PAR-1 or PAR-2 deficient mice, (c) and following in vivo activation of PARs. FX knockout mice with residual activity of only 1–3% of normal (n=9) were injected with AAV-2-human(h)FIX vector and compared to littermates with FX levels of 50% (n=4). FIX expression levels were 2-fold lower among FX-deficient mice compared to controls (p<0.03). The second model, FIX deficient mice, received AAV expressing α1-antitrypsin (AAT-1). Severe hemophilia B models due to large-gene deletion (n=5) or missense mutation (R180T) in the FIX gene (n=3, <1% FIX) were compared to littermate controls with normal FIX levels (n=6). The results showed that AAT-1 levels among hemophilia B mice were 2-fold lower than in controls (24 vs 48 ng/ml, p<0.05, respectively). Because PAR activation by thrombin enhances αVβ5 (co-receptor for AAV-2 and adenovirus)-dependent cellular function (JBC 276:10952) we hypothesized that PAR modulates AAV-2 gene transfer. Homozygous (−/−) or heterozygous deficient (+/−) PAR-1 (n=24) or PAR-2 (n=25) mice received AAV-2-hF.IX and were compared to littermate controls (+/+). FIX levels among PAR-1 controls (1.9 μg/ml) were comparable to levels obtained among heterozygotes but higher than in homozygotes (1.1 μg/ml, p<0.02). Similarly, PAR-2 deficient mice presented 2-fold lower FIX levels than controls (0.7 vs 1.3 μg/ml, p<0.02) whereas heterozygous mice presented intermediate levels. To further confirm the role of PARs in AAV-2 gene transfer we activated PARs prior to AAV-2 injection. C57BL/6 mice received specific peptide agonists at doses ranging from 10 to 60 μM/kg (n=4 per dose and per peptide) and were compared to controls receiving scramble peptide. FIX levels increased 1.5 to 5-fold in a dose-dependent manner and the activation of PAR-1 and -2 simultaneously was superior to single peptide. Gene copy monitoring revealed low vector uptake by livers of PAR knockout mice while activation of PARs increased uptake. In conclusion, these data demonstrated a novel in vivo role of coagulation proteases and PARs on viral vectors (AAV-2 and adenovirus)-mediated gene expression and provide an alternative target to modulate gene therapy strategies.

Abstract 5290: Cypher Long but not Short Isoform Specific Knockout Mice Result in Cardiac Signaling Defects and Dilated Cardiomyopathy

Circulation ◽  
2008 ◽  
Vol 118 (suppl_18) ◽  
Author(s):  
Hongqiang Cheng ◽  
Ming Zheng ◽  
Farah Sheikh ◽  
Kunfu Ouyang ◽  
Li Cui ◽  
...  
Keyword(s):  
Dilated Cardiomyopathy ◽  
Knockout Mice ◽  
Molecular Mechanisms ◽  
Heart Function ◽  
Gene Mutations ◽  
Splice Isoforms ◽  
Functional Roles ◽  
Deficient Mice

Our previous studies have demonstrated that Cypher, a PDZ-LIM protein localized at the Z line, plays a pivotal role in heart function. We recently identified long and short splice isoforms of Cypher, which are characterized by the presence and absence of LIM domains, respectively. The LIM domain of Cypher is thought to be involved in signaling, based on its ability to directly interact with signaling proteins. In human patients with dilated cardiomyopathy (DCM) we discovered Cypher gene mutations, which affect either long or short isoform or both isoforms. However, the precise molecular mechanisms underlying the role of Cypher isoforms in DCM remain unclear. To determine the role of Cypher isoforms in cardiac signaling and disease in vivo , we generated two Cypher isoform specific knockout mice. Selective ablation of Cypher long isoforms in mice resulted in partial neonatal lethality. However, hearts from viable Cypher long isoform deficient mice displayed Z line abnormalities and decreased cardiomyocyte widths, which resulted in a progressive form of DCM, characterized by fibrosis, calcification and lethality. The effects on cardiac function and disease observed in long-isoform specific Cypher knockout mice were preceded by significant decreases in cardiac protein kinase C and extracellular signal-regulated kinase signaling. These results are in contrast to Cypher short isoform deficient mice, which were viable with no overt cardiac morphology and signaling abnormalities. These results reveal distinct functional roles for Cypher isoforms in the heart as well as shed light into the molecular mechanisms underlying dilated cardiomyopathy.

scholarly journals Mice Deficient for the Ets Transcription Factor Elk-1 Show Normal Immune Responses and Mildly Impaired Neuronal Gene Activation

2004 ◽  
Vol 24 (1) ◽  
pp. 294-305 ◽  
Author(s):  
Francesca Cesari ◽  
Stephan Brecht ◽  
Kristina Vintersten ◽  
Lam Giang Vuong ◽  
Matthias Hofmann ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Transcriptional Activation ◽  
Embryonic Stem ◽  
Adult Life ◽  
Mitogen Activated Protein Kinase ◽  
Wild Type ◽  
Mouse Development ◽  
Serum Response ◽  
Deficient Mice

ABSTRACT The transcription factor Elk-1 belongs to the ternary complex factor (TCF) subfamily of Ets proteins. TCFs interact with serum response factor to bind jointly to serum response elements in the promoters of immediate-early genes (IEGs). TCFs mediate the rapid transcriptional response of IEGs to various extracellular stimuli which activate mitogen-activated protein kinase signaling. To investigate physiological functions of Elk-1 in vivo, we generated Elk-1-deficient mice by homologous recombination in embryonic stem cells. These animals were found to be phenotypically indistinguishable from their wild-type littermates. Histological analysis of various tissues failed to reveal any differences between Elk-1 mutant and wild-type mice. Elk-1 deficiency caused no changes in the proteomic displays of brain or spleen extracts. Also, no immunological defects could be detected in mice lacking Elk-1, even upon infection with coxsackievirus B3. In mouse embryonic fibroblasts, Elk-1 was dispensable for c-fos and Egr-1 transcriptional activation upon stimulation with serum, lysophosphatidic acid, or tetradecanoyl phorbol acetate. However, in brains of Elk-1-deficient mice, cortical and hippocampal CA1 expression of c-fos, but not Egr-1 or c-Jun, was markedly reduced 4 h following kainate-induced seizures. This was not accompanied by altered patterns of neuronal apoptosis. Collectively, our data indicate that Elk-1 is essential neither for mouse development nor for adult life, suggesting compensatory activities by other TCFs.

scholarly journals Lessons from Tau-Deficient Mice

2012 ◽  
Vol 2012 ◽  
pp. 1-8 ◽  
Author(s):  
Yazi D. Ke ◽  
Alexandra K. Suchowerska ◽  
Julia van der Hoven ◽  
Dineeka M. De Silva ◽  
Christopher W. Wu ◽  
...  
Keyword(s):  
Knockout Mice ◽  
Mouse Strains ◽  
Motor Deficits ◽  
Transgenic Mouse Models ◽  
The Past ◽  
Number Of Publications ◽  
The Brain ◽  
Deficient Mice

Both Alzheimer's disease (AD) and frontotemporal dementia (FTD) are characterized by the deposition of hyperphosphorylated forms of the microtubule-associated protein tau in neurons and/or glia. This unifying pathology led to the umbrella term “tauopathies” for these conditions, also emphasizing the central role of tau in AD and FTD. Generation of transgenic mouse models expressing human tau in the brain has contributed to the understanding of the pathomechanistic role of tau in disease. To reveal the physiological functions of tauin vivo, several knockout mouse strains with deletion of the tau-encodingMAPTgene have been established over the past decade, using different gene targeting constructs. Surprisingly, when initially introduced tau knockout mice presented with no overt phenotype or malformations. The number of publications using tau knockout mice has recently markedly increased, and both behavioural changes and motor deficits have been identified in aged mice of certain strains. Moreover, tau knockout mice have been instrumental in identifying novel functions of tau, both in cultured neurons andin vivo. Importantly, tau knockout mice have significantly contributed to the understanding of the pathophysiological interplay between Aβand tau in AD. Here, we review the literature that involves tau knockout mice to summarize what we have learned so far from depleting tauin vivo.

scholarly journals Naa12 rescues embryonic lethality in Naa10-Deficient Mice in the amino-terminal acetylation pathway

2020 ◽  
Author(s):  
Hyae Yon Kweon ◽  
Mi-Ni Lee ◽  
Max Dörfel ◽  
Seungwoon Seo ◽  
Leah Gottlieb ◽  
...  
Keyword(s):  
Embryonic Lethality ◽  
Mouse Development ◽  
Post Translational Modifications ◽  
Amino Terminal ◽  
Neonatal Lethality ◽  
Enormous Amount ◽  
Deficient Mice ◽  
Urogenital Anomalies

AbstractThere is an enormous amount of variation in proteins introduced by co- and post-translational modifications, including N-terminal acetylation (NTA), catalyzed by a set of N-terminal acetyltransferases (NATs). The NatA complex (including X-linked Naa10 and Naa15) is the major acetyltransferase, with 40–50% of all mammalian proteins being potential substrates. However, the overall role of NTA on a whole-organism level is poorly understood, particularly in mammals. Male mice lacking Naa10 show no globally apparent in vivo NTA impairment and, surprisingly, do not exhibit embryonic lethality. Rather Naa10 nulls display increased neonatal lethality, and the majority of surviving undersized mutants exhibit a combination of hydrocephaly, cardiac defects, homeotic anterior transformation (including an extra thoracic rib), piebaldism and urogenital anomalies. The lack of complete embryonic lethality in Naa10-null mice is explained by the discovery of Naa12, a previously unannotated Naa10-like paralogue with NAT activity that genetically compensates for Naa10. Mice deficient for Naa12 have no apparent phenotype, except for decreased fertility, whereas mice doubly deficient for Naa10 and Naa12 display embryonic lethality, thus presenting the complete machinery for NatA-mediated NTA in mouse development.

scholarly journals Functions of paralogous RNA polymerase III subunits POLR3G and POLR3GL in mouse development

2020 ◽  
Vol 117 (27) ◽  
pp. 15702-15711
Author(s):  
Xiaoling Wang ◽  
Alan Gerber ◽  
Wei-Yi Chen ◽  
Robert G. Roeder
Keyword(s):  
Rna Polymerase ◽  
Knockout Mice ◽  
Mammalian Cells ◽  
Target Genes ◽  
Rna Polymerase Iii ◽  
Embryonic Stem ◽  
Mouse Development ◽  
Polymerase Iii ◽  
Pol Iii

Mammalian cells contain two isoforms of RNA polymerase III (Pol III) that differ in only a single subunit, with POLR3G in one form (Pol IIIα) and the related POLR3GL in the other form (Pol IIIβ). Previous research indicates that POLR3G and POLR3GL are differentially expressed, with POLR3G expression being highly enriched in embryonic stem cells (ESCs) and tumor cells relative to the ubiquitously expressed POLR3GL. To date, the functional differences between these two subunits remain largely unexplored, especially in vivo. Here, we show that POLR3G and POLR3GL containing Pol III complexes bind the same target genes and assume the same functions both in vitro and in vivo and, to a significant degree, can compensate for each other in vivo. Notably, an observed defect in the differentiation ability of POLR3G knockout ESCs can be rescued by exogenous expression of POLR3GL. Moreover, whereas POLR3G knockout mice die at a very early embryonic stage, POLR3GL knockout mice complete embryonic development without noticeable defects but die at about 3 wk after birth with signs of both general growth defects and potential cerebellum-related neuronal defects. The different phenotypes of the knockout mice likely reflect differential expression levels of POLR3G and POLR3GL across developmental stages and between tissues and insufficient amounts of total Pol III in vivo.

scholarly journals Dimerization of CUL7 and PARC Is Not Required for All CUL7 Functions and Mouse Development

2005 ◽  
Vol 25 (13) ◽  
pp. 5579-5589 ◽  
Author(s):  
Jeffrey R. Skaar ◽  
Takehiro Arai ◽  
James A. DeCaprio
Keyword(s):  
Knockout Mice ◽  
Cytoplasmic Protein ◽  
Mouse Development ◽  
Interacting Protein ◽  
Targeted Deletion ◽  
Neonatal Lethality ◽  
The Stability ◽  
And Function

ABSTRACT CUL7, a recently identified member of the cullin family of E3 ubiquitin ligases, forms a unique SCF-like complex and is required for mouse embryonic development. To further investigate CUL7 function, we sought to identify CUL7 binding proteins. The p53-associated, parkin-like cytoplasmic protein (PARC), a homolog of CUL7, was identified as a CUL7-interacting protein by mass spectrometry. The heterodimerization of PARC and CUL7, as well as homodimerization of PARC and CUL7, was confirmed in vivo. To determine the biological role of PARC by itself and in conjunction with CUL7, a targeted deletion of Parc was created in the mouse. In contrast to the neonatal lethality of the Cul7 knockout mice, Parc knockout mice were born at the expected Mendelian ratios and exhibited no apparent phenotype. Additionally, Parc deletion did not appear to affect the stability or function of p53. These results suggest that PARC and CUL7 form an endogenous complex and that PARC and CUL7 functions are at least partially nonoverlapping. In addition, although PARC and p53 form a complex, the absence of effect of Parc deletion on p53 stability, localization, and function suggests that p53 binding to PARC may serve to control PARC function.

scholarly journals Sox12 Deletion in the Mouse Reveals Nonreciprocal Redundancy with the Related Sox4 and Sox11 Transcription Factors

2008 ◽  
Vol 28 (15) ◽  
pp. 4675-4687 ◽  
Author(s):  
Melanie Hoser ◽  
Michaela R. Potzner ◽  
Julia M. C. Koch ◽  
Michael R. Bösl ◽  
Michael Wegner ◽  
...  
Keyword(s):  
Transcription Factors ◽  
B Cell Development ◽  
Mouse Development ◽  
Expression Levels ◽  
Binding Characteristics ◽  
Sox Proteins ◽  
Deficient Mice

ABSTRACT The transcription factors Sox4 and Sox11 are important regulators of diverse developmental processes including heart, lung, pancreas, spleen, and B-cell development. Here we have studied the role of the related Sox12 as the third protein of the SoxC group both in vivo and in vitro. Despite widespread Sox12 expression during embryonic development, Sox12-deficient mice developed surprisingly normally, so that they were born alive, showed no gross phenotypic abnormalities, and were fertile in both sexes. Comparison with the related Sox4 and Sox11 revealed extensive overlap in the embryonic expression pattern but more uniform expression levels for Sox12, without sites of particularly high expression. All three Sox proteins furthermore exhibited comparable DNA-binding characteristics and functioned as transcriptional activators. Sox12 was, however, a relatively weak transactivator in comparison to Sox11. We conclude that Sox4 and Sox11 function redundantly with Sox12 and can compensate its loss during mouse development. Because of differences in expression levels and transactivation rates, however, functional compensation is not reciprocal.

scholarly journals Normal Hematopoiesis and Inflammatory Responses Despite Discrete Signaling Defects in Gα15 Knockout Mice

2000 ◽  
Vol 20 (3) ◽  
pp. 797-804 ◽  
Author(s):  
I. Davignon ◽  
M. D. Catalina ◽  
D. Smith ◽  
J. Montgomery ◽  
J. Swantek ◽  
...  
Keyword(s):  
Knockout Mice ◽  
Inflammatory Responses ◽  
Trichinella Spiralis ◽  
Primary Cultures ◽  
Hematopoietic Cells ◽  
Embryonic Stem ◽  
Double Knockout ◽  
C5a Receptor ◽  
Α Subunits

ABSTRACT Gα15 activates phospholipase Cβ in response to the greatest variety of agonist-stimulated heptahelical receptors among the four Gq class G-protein α subunits expressed in mammals. Gα15 is primarily expressed in hematopoietic cells in fetal and adult mice. We disrupted the Gα15 gene by homologous recombination in embryonic stem cells to identify its biological functions. Surprisingly, hematopoiesis was normal in Gα15−/− mice, Gα15−/−Gαq−/− double-knockout mice (which express only Gα11 in most hematopoietic cells), and Gα11−/− mice, suggesting functional redundancy in Gq class signaling. Inflammatory challenges, including thioglycolate-induced peritonitis and infection with Trichinella spiralis, stimulated similar responses in Gα15−/− adults and wild-type siblings. Agonist-stimulated Ca2+ release from intracellular stores was assayed to identify signaling defects in primary cultures of thioglycolate-elicited macrophages isolated from Gα15−/− mice. C5a-stimulated phosphoinositide accumulation and Ca2+ release was significantly reduced in Gα15−/− macrophages. Ca2+ signaling was abolished only in mutant cells pretreated with pertussis toxin, suggesting that the C5a receptor couples to both Gα15 and Gαi in vivo. Signaling evoked by other receptors coupled by Gq class α subunits appeared normal in Gα15−/− macrophages. Despite discrete signaling defects, compensation by coexpressed Gq and/or Gi class α subunits may suppress abnormalities in Gα15-deficient mice.

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